The had already reached a certain extent before

The colonization of the zinnia roots
by all AM treatments was high and only the highest level of DS could reduce it
(Table 1). Negative effect of DS on mycorrhizal colonization has been reported
by many researchers (Fagbola et al. 2001).
However, in our experiment, Low effect of drought stress on mycorrhizal
colonization may have been related to insufficient time of exposure to the
drought because colonization rate had already reached a certain extent before
the drought treatment was applied. It seems that once the frequency of
mycorrhizal colonization reached a certain extent it was not easy to affect it
by a short-term stress.

Although +M zinnia seedlings
accumulated more biomass under DS as well as well-watered condition compared
with the -M ones, drought had a marginal effect on root dry weight.
Furthermore, biomass accumulation of -M plants were not affected by any of
drought levels. The reason might be that biomass accumulation is a long-term
process for plant, thus the short-term drought period may not greatly influence
the biomass.

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Plant biomass production is directly
linked to photosynthesis, the most important anabolic processes of higher
plants that is very sensitive to DS (Mo et al.
2016). The results of this research supported this sensitivity, i.e., Pn
of zinnia seedlings was reduced considerably under DS conditions;
however, the AM symbiosis could mitigate the negative effects to some extent.
In this experiment, +M zinnia plants showed higher Pn, gs,
E, WUEi and lower Ci (Table 2).
These findings are in good agreement with previous studies (Porcel et al. 2015). It has been suggested that
the drought-induced suppression of photosynthesis could be generally attributed
to stomatal limitation and/or non-stomatal/metabolic limitation. Some
researchers agree that the stomatal closure and resulting CO2
deficit is the main cause of decreased photosynthesis under mild and moderate DS
(Flexas and Medrano 2002) while some
believe that gs only affects Pn at severe DS
(Chaves and Oliveira 2004). The linear correlation
between Pn and gs showed that although the
relationship between Pn and gs was
decreased with increasing drought stress intensity, but it was still
significant under FC40 (r=0.55) indicated that reduction in Pn
under severe DS conditions was regulated mostly by stomatal factors (Table 5).
Irrespective of AM treatments, DS increased Ci at FC60
and FC40 and reduced E at FC40. Our results
regarding the effect of DS on the Ci and E were in agreement
with previous works (Abid et al. 2016).
The increase in Ci in drought stressed zinnias indicates the role of
non-stomatal limitations (mesophyll resistance) to photosynthesis. A high Ci
value associated with a low stomatal conductance would indicate a decrease in
the Pn/Ci ratio which can be considered the
estimate of Rubisco activity, illustrating its limitations under stress
conditions (Niinemets et al. 2016).
Reduced transpiration under severe DS could be attributed to decrease in
stomatal conductance or stomatal density, however, a significant correlation
between gs and E was found in all drought stress
levels (Table 5). Higher gs and E in +M compared to -M
plants may indicate lower resistance to water vapor transfer from inside the
leaves to the atmosphere, which allows leaves to maintain more normal water
balance. A reduction of WUEi is observed, especially for
stressed plants. This result is in agreement with others showing that under
severe stress conditions intrinsic water use efficiency (WUEi)
decreases (Dias and Brüggemann 2010).
Contrary to WUEi, WUEp increased in
response to drought. This change is considered a key factor determining plant
productivity under limited water supply and it is mentioned as an adaptive
response and a strategy to improve crop performance under limited water
conditions (Chaves et al. 2009). AM
treatments significantly increased the WUEp under the highest
DS level (FC40) in compare with respected control. The positive
effect of AM symbiosis on WUEp under drought stress have been
reported in previous studies (Kaya et al. 2003)
showing +M plants are more successful in terms of dry matter
production and water use than the –M plants.

PSII complex is the most sensitive
component of the electron transport chain to environmental stresses. Several
studies have demonstrated damage to the PSII oxygen-evolving complex and the
PSII reaction centers, and in turn, degradation of D1 protein under DS (Maxwell and Johnson 2000). Our results showed
that irrespective of mycorrhizal treatments, drought induced a reduction in Fm,
Fv, Fv/Fm, Fv/F0 and
PIABS while it had no significant effect on F0.
The reduction was relatively higher in proportion with drought intensity. On
the other hand, in most of DS levels, +M zinnia plants showed higher Fv/Fm,
Fv/F0, Fv, Fm, PIABS
and lower F0 than –M zinnias. This indicates that an AM
symbiotic relationship can enhance the efficiency of excitation energy capture
by chloroplasts, increase the photochemical capacity of PSII. Increments in F0
values associated with reductions in Fm values can be
interpreted as an indication of the damage in the light-harvesting complex of
PSII (Li et al. 2010), as observed in the
present study in the case of –M plants. As the effect of drought, Fv/Fm
was decreased in FC60 and FC40. Fv/Fm
in all +M zinnia plants was higher than in -M plants regardless of drought
treatments. Photoinhibition is represented by decreasing Fv/Fm
ratio (Piper et al. 2007). Decreases of
the Fv/Fm ratio may be the result of Calvin cycle
disturbances that delay redoxidation of QA- and induce
photosystem II down-regulation or damage thylakoid membrane electron transport
(Galle et al. 2002). We studied the
correlations between photosynthesis and the Fv/Fm
ratio in zinnia seedlings. There were significant correlations between the CO2
assimilation rate and the Fv/Fm ratio in all
DS levels (Table 5).At the same time, the parameter Fv/F0
was also higher in +M plants. The Fv/F0 ratio is a
very sensitive indicator of the potential photosynthetic activity, in both
stressed and healthy plants (Maxwell and Johnson
2000). These results indicated that AM symbioses might improve
photochemistry efficiency of PSII under well-watered and droughted conditions. PIABS
reflects the functionality of both photosystems I and II and gives us
quantitative information on the current state of plant performance under stress
conditions. Many authors confirm that plant’s first reaction to drought stress
is increase in photosynthetic efficiency parameters and thus performance index
as well (Kovacevic et al. 2013). In
our experiment, Fv/Fm better reflected expected
changes in leaf photosynthetic performance under different DS levels whereas no
correlation was found between Pn and PIABS
in FC40 (Table 5).

Drought has a significant effect on
chlorophyll content, especially chlorophyll a. This decrease has been explained
by some authors as a photoprotection mechanism through reducing light
absorbance (Abid et al. 2016). On the
other hand, chlorophyll concentration has been known as an index for evaluation
of source (Zobayed et al. 2005),
therefore decrease of this can be considered as a non-stomata limiting factor
in the drought stress conditions. In this experiment, DS reduced chlorophyll
concentration at FC60 and FC40 and there was no
correlation between Pn and Chl at these levels of DS
(Table 5) showing other factors than that of Chl content are responsible
for decreasing Pn under severe DS in zinnia plants. Our
results showed that irrespective of DS, +M zinnia plants had higher Chl
content (a,b,a+b), support previous findings that AM fungi inoculation
enhances the Chl content of their host plant (Mo et al. 2016) and so induces drought tolerance.

In this comparative study, three
fungal species were studied singly or in combination for their ability to
enhance the drought tolerance of zinnia plants. The ranking of AMF effects on
drought tolerance, based on the decreases in SDW, was as follows: Funneliformis
mosseae>Rhizophagus intraradices>Rhizophagus irregularis>AMF
mixture. As it is evident, the mixed inoculation did not enhance
growth-promotion effects although it had the highest rate of colonization
(Table 1). Therefore, the promoting effects of the mycorrhizal isolates were
not associated with colonizing ability. This result may be explained as that
one of the inoculated species was not effective for the plant. Some reports
indicated that maximum bene?ts to plants might be achieved with a single, most
ef?cient AMF species, and indicated that mycorrhizal diversity would not bring
further bene?ts (Edathil et al. 1996).
Furthermore, high effectiveness of F. mosseae in this experiment was in
accordance with previous work in which G. mosseae was introduced as the
most effective species for zinnia (Long et al.
2010). All AM treatments produced considerably more SDW and RDW under
non-stress condition (FC100) in compare with respected –M control
showing the beneficial effects of this symbiosis even in normal condition.

In conclusion, this study was
achieved to investigate the potential for improving drought tolerance of zinnia
(Zinnia elegans var. Magellan Red) by different AM species. Zinnia
plants benefited from all mycorrhizal associations. Results showed the –M
zinnia plants to be the most affected by stress conditions as it exhibited the
lowest values for SDW and RDW resulted from a significant decline in Pn,
gs, E, Fv/Fm, PIABS, Chl
content, WUE in compare with +M plants. Such results indicate the
improved performance of the photosynthetic machinery and the absence of
photoinhibition when +M plants were exposed to water deficit as it was obvious
from strong correlations between Pn and AM
colonization under all DS levels.